Phosphatidylinositol:myo-Inositol Exchange Activity in Intact Nerve Endings: Substrate and Cofactor Dependence, Nucleotide Specificity, and Effect on Synaptosomal Handling of myo-Inositol

Micromolar concentrations of CMP produced a large increase in Mn2+-dependent phosphatidylinositol:myo-inositol exchange activity in isolated nerve endings or synaptosomes. The apparent Km for CMP was 2 microM, and that for myo-inositol was 38 microM. Only cytidine nucleotides were capable of enhancing activity, and this effect is probably specific for CMP, because the synaptosomal preparation rapidly converted CTP or CDP to CMP. Manganese did not affect the uptake of myo-inositol into the synaptosomal cytosolic fraction or myo-inositol levels. Determinations of myo-inositol specific activity showed that the Mn2+-enhanced labeling of phosphatidylinositol was not accompanied by a decrease of label content in free myo-inositol. This lack of an effect on intrasynaptosomal myo-inositol and the dependence of exchange on cytidine nucleotides whereas cytidine itself was previously found to be without effect show that for the bulk of Mn2+-dependent exchange activity, it is the myo-inositol in the incubation medium that is being directly incorporated into membrane-bound phosphatidyl-inositol. Because CMP dependence is the hallmark of exchange catalyzed by CDP-diacylglycerol:inositol phosphatidyl transferase, this enzyme is likely to be responsible for most of the exchange activity in synaptosomes. The strong affinity of this exchange system for CMP suggests that endogenous levels of this nucleotide might support Mn2+-dependent exchange in the absence of added nucleotide.     

Acute and Subacute Effects of Thiamine Deficiency On the Morphometry and Cell Kinetics of Jejunal and Ileal Epithelial Cells

Abstract. The effects of acute and subacute thiamine deficiency on jejunal and ileal epithelial cells were studied in rats, using crypt and villus cell population, crypt cell production per crypt (CCPC), crypt growth fraction (I_p) and crypt cell cycle time (T_c) as parameters. In acute thiamine deficiency there was marked jejunal hypoplasia of the crypt and villus, but in the ileum there was hypoplasia only of the crypt. the jejunal epithelium of the subacute thiamine deficiency (STD) group showed no morphometric changes. In contrast, in the ileal epithelium of STD rats there was decreased crypt depth and villus cell population. Thiamine deficiency had no significant effect on CCPC, I_p and T_c.     

Acute and subacute effects of thiamine deficiency on the morphometry and cell kinetics of jejunal and ileal epithelial cells

The effects of acute and subacute thiamine deficiency on jejunal and ileal epithelial cells were studied in rats, using crypt and villus cell population, crypt cell production per crypt (CCPC), crypt growth fraction (Ip) and crypt cell cycle time (Tc) as parameters. In acute thiamine deficiency there was marked jejunal hypoplasia of the crypt and villus, but in the ileum there was hypoplasia only of the crypt. The jejunal epithelium of the subacute thiamine deficiency (STD) group showed no morphometric changes. In contrast, in the ileal epithelium of STD rats there was decreased crypt depth and villus cell population. Thiamine deficiency had no significant effect on CCPC, Ip and Tc.     

Recombinant interleukin-4-treated macrophages, epithelioid cell surrogates, harbor and arrest Mycobacterium avium multiplication in vitro

Our group has previously described that murine peritoneal macrophages treated in vitro for 7 days with recombinant interleukin-4 (rIL-4) acquire morphological and functional characteristics of epithelioid cells (ECs) found in granulomatous lesions. Although EC function has not been clarified so far, it has been suggested that these cells could present antigens and control multiplication of mycobacteria. These aspects have been addressed here using in vitro EC surrogates. Using immunocytochemistry and immunofluorescence methods, we have observed an increased expression of CD11b, CD54, CD86 and CD40 molecules on rIL-4-treated macrophages when compared to untreated ones. Cytokine-treated cells were less phagocytic for latex beads (P<0.03) and more pinocytic for dextran particles than untreated macrophages. T-cell lymphoproliferation assays using ovalbumin (OVA) and Mycobacterium avium as antigens showed that both cultured macrophages were equally efficient as antigen presenting cells (APCs). However, M. avium antigens were better presented in vivo by EC surrogates (P<0.01). Both macrophage cultures were similarly infected by M. avium. However, while the infection level was maintained in the cytokine-treated population, untreated macrophages showed a progressive increase in the number of bacilli/cell with time (P<0.01) and a reduction of about 65% in cell population. After 96 h of M. avium infection, untreated cells secreted higher amounts of tumor necrosis factor-alpha (P<0.005) while rIL-4-treated macrophages showed higher, although not significant, transforming growth factor-beta production. Also, EC surrogates produced less nitric oxide than control macrophages (P<0.05). Hence, EC surrogates restrain M. avium growth and act as APCs in vitro and in vivo.     

Mutations in the dimerization domain of the b subunit from the Escherichia coli ATP synthase. Deletions disrupt function but not enzyme assembly

The b subunit dimer of Escherichia coli ATP synthase serves essential roles as an assembly factor for the enzyme and as a stator during rotational catalysis. To investigate the functional importance of its coiled coil dimerization domain, a series of internal deletions including each individual residue between Lys-100 and Ala-105 (b(deltaK100)-b(deltaA105)), b(deltaK100-A103), and b(deltaK100-Q106) as well as a control b(K100A) missense mutation were prepared. All of the mutants supported assembly of ATP synthase, but all single-residue deletions failed to support growth on acetate, indicating a severe defect in oxidative phosphorylation, and b(deltaK100-Q106) displayed moderately reduced growth. The membrane-bound ATPase activities of these strains showed a related reduction in sensitivity to dicyclohexylcarbodiimide, indicative of uncoupling. Analysis of dimerization of the soluble constructs of b(deltaK100) and the multiple-residue deletions by sedimentation equilibrium revealed reduced dimerization compared with wild type for all deletions, with b(deltaK100-Q106) most severely affected. In cross-linking studies it was found that F1-ATPase can mediate the dimerization of some soluble b constructs but did not mediate dimerization of b(deltaK100) and b(deltaK100-Q106); these two forms also were defective in F1 binding analyses. We conclude that defective dimerization of soluble b constructs severely affects F1 binding in vitro, yet allows assembly of ATP synthase in vivo. The highly uncoupled nature of enzymes with single-residue deletions in b indicates that the b subunit serves an active function in energy coupling rather than just holding on to the F1 sector. This function is proposed to depend on proper, specific interactions between the b subunits and F1.     

Stochastic high-speed rotation of Escherichia coli ATP synthase F1 sector: the epsilon subunit-sensitive rotation

The gamma subunit of the ATP synthase F(1) sector rotates at the center of the alpha(3)beta(3) hexamer during ATP hydrolysis. A gold bead (40-200 nm diameter) was attached to the gamma subunit of Escherichia coli F(1), and then its ATP hydrolysis-dependent rotation was studied. The rotation speeds were variable, showing stochastic fluctuation. The high-speed rates of 40- and 60-nm beads were essentially similar: 721 and 671 rps (revolutions/s), respectively. The average rate of 60-nm beads was 381 rps, which is approximately 13-fold faster than that expected from the steady-state ATPase turnover number. These results indicate that the F(1) sector rotates much faster than expected from the bulk of ATPase activity, and that approximately 10% of the F(1) molecules are active on the millisecond time scale. Furthermore, the real ATP turnover number (number of ATP molecules converted to ADP and phosphate/s), as a single molecule, is variable during a short period. The epsilon subunit inhibited rotation and ATPase, whereas epsilon fused through its carboxyl terminus to cytochrome b(562) showed no effect. The epsilon subunit significantly increased the pausing time during rotation. Stochastic fluctuation of catalysis may be a general property of an enzyme, although its understanding requires combining studies of steady-state kinetics and single molecule observation.     

Ultrasonic pregnancy diagnosis in the camel

Application of ultrasound for pregnancy diagnosis has been tested and evaluated in 15 Iranian camels (Camelus dromedarius), all of which ultimately calved. Transabdominal examinations were unsuccessful, while intrapelvic application resulted in the reception of sounds characteristic for foetal life, similar to those found in other domestic animals. Signals of foetal heart, pulse of umbilical vessels and uterine artery as well as foetal movement could be recognized as distinct sounds and have been recorded for further studies. An attempt was made to verify the findings of the ultrasonic diagnosis through rectal palpation. The ultrasonic technique resulted in 12 correct and three incorrect diagnoses.     

Molecular evolution of the mammalian ribosomal protein gene, RPS14

Ribosomal protein S14 genes (RPS14) in eukaryotic species from protozoa to primates exhibit dramatically different intron-exon structures yet share homologous polypeptide-coding sequences. To recognize common features of RPS14 gene architectures in closely related mammalian species and to evaluate similarities in their noncoding DNA sequences, we isolated the intron-containing S14 locus from Chinese hamster ovary (CHO) cell DNA by using a PCR strategy and compared it with human RPS14. We found that rodent and primate S14 genes are composed of identical protein-coding exons interrupted by introns at four conserved DNA sites. However, the structures of corresponding CHO and human RPS14 introns differ significantly. Nonetheless, individual intron splice donor, splice acceptor, and upstream flanking motifs have been conserved within mammalian S14 homologues as well as within RPS14 gene fragments PCR amplified from other vertebrate genera (birds and bony fish). Our data indicate that noncoding, intronic DNA sequences within highly conserved, single-copy ribosomal protein genes are useful molecular landmarks for phylogenetic analysis of closely related vertebrate species.      

How to contend with paraphyly in the taxonomy of the delphinine cetaceans?

Molecular phylogenetic analyses conducted over the past 15 yr have consistently had difficulties resolving relationships among the cetacean species in the subfamily Delphininae. In addition, paraphyly of the genera Tursiops and Stenella in these molecular phylogenies has been a recurrent problem since the first appearance of such a phylogeny in 1999, suggesting that these genera do not accurately reflect the evolutionary relationships of the species they contain. Morphological analyses have not resolved the issues. The genera in Delphininae originated in the 19th Century on questionable morphological grounds. The species were nearly all originally described in the genus Delphinus of Linnaeus. Recent molecular phylogenies based on various mitochondrial and nuclear DNA markers have suggested a wide range of possible relationships among these taxa, and several authors have suggested synonymizing all the taxa (Lagenodelphis, Stenella, Sousa, and Tursiops) under Delphinus. Until molecular and/or morphological analyses adequately sort out relationships in this very recently radiated group, one possible solution indeed would be to merge all the delphinine genera with Delphinus. Implications of such a move and alternatives are discussed.

Editor's Note: Papers from past Norris Award winners have primarily been a revised or reduced version of the actual presentation given as a plenary talk at the biennial conference. Dr. Perrin requested being allowed to take a topic from his presentation and expand on it to present a set of ideas in the form of an essay that could pass the rigors of the peer‐review process. As a result, this Norris Award paper has undergone peer‐review and has taken longer than usual for a Norris Award paper to appear in the journal following its presentation at the biennial conference. It also has co‐authors, with varying opinions on the issues discussed in the essay, to cover appropriately and more thoroughly those components of the paper that required additional expertise. I believe this approach has produced an excellent, thought‐provoking essay and is an approach that should be available to future Norris Award winners if they so choose to take it. Since this essay is meant to elicit dialogue, comments are welcome and will be considered for publication in Letters to the Editor.